Use of rna for reprogramming somatic cells

ABSTRACT

Methods for effecting the de-differentiation of somatic cells to cells having stem cell characteristics, in particular pluripotency, include the steps of introducing RNA encoding factors inducing the de-differentiation of somatic cells into the somatic cells and culturing the somatic cells allowing the cells to de-differentiate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/250,366 filed on Jan. 17, 2019, which in turn is a continuation ofU.S. patent application Ser. No. 15/485,601, filed on Apr. 12, 2017,which is a continuation of U.S. patent application Ser. No. 14/933,840,filed on Nov. 5, 2015, which is a continuation of U.S. patentapplication Ser. No. 12/735,060, filed on Nov. 24, 2010, now abandoned,which is the National Stage of International Patent Application No.PCT/EP2008/010593, filed on Dec. 12, 2008, which claims priority ofEuropean Patent Application No. 07024312.6, filed on Dec. 14, 2007, eachof which is incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

This application includes biological sequence information, which is setforth in an ASCII text file having the file name“VOS-128-CON-3_SEQ.txt”, created on Jan. 17, 2019, and having a filesize of 32,591 bytes, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides methods for de-differentiating somaticcells into stem-like cells without generating embryos or fetuses. Morespecifically, the present invention provides methods for effecting thede-differentiation of somatic cells to cells having stem cellcharacteristics, in particular pluripotency, by introducing RNA encodingfactors inducing the de-differentiation of somatic cells into thesomatic cells and culturing the somatic cells allowing the cells tode-differentiate. After being de-differentiated, the cells can beinduced to re-differentiate into the same or a different somatic celltype such as neuronal, hematopoietic, muscle, epithelial, and other celltypes. The stem-like cells derived by the present invention have medicalapplications for treatment of degenerative diseases by “cell therapy”and may be utilized in novel therapeutic strategies in the treatment ofcardiac, neurological, endocrinological, vascular, retinal,dermatological, muscular-skeletal disorders, and other diseases.

BACKGROUND OF THE INVENTION

Stem cells also called progenitor cells are cells with abilities toself-renew, to remain undifferentiated, and to become differentiatedinto one or more specialized cell types with mature phenotypes. Stemcells are not terminally differentiated and they are not at the end of adifferentiation pathway.

Totipotent cells contain all the genetic information needed to createall the cells of the body, including the cells of the placenta. Humancells have this totipotent capacity only during the first few divisionsof a fertilized egg. After three to four divisions of totipotent cells,there follows a series of stages in which the cells become increasinglyspecialized. The next stage of division results in pluripotent cells,which are highly versatile and can give rise to any cell type except thecells of the placenta or other supporting tissues of the uterus. At thenext stage, cells become multipotent, meaning they can give rise toseveral other cell types, but those types are limited in number. At theend of the long chain of cell divisions that make up the embryo are“terminally differentiated” cells that are considered to be permanentlycommitted to a specific function.

There are three main groups of stem cells: (i) adult or somatic stemcells (post-natal), which exist in all post-natal organisms, (ii)embryonic stem cells, which can be derived from a pre-embryonic orembryonic developmental stage and (iii) fetal stem cells (pre-natal),which can be isolated from the developing fetus.

Stem cell technologies involving the isolation and use of humanembryonic stem cells have become an important subject of medicalresearch. Human embryonic stem cells have a potential to differentiateinto any and all of the cell types in the human body, including complextissues. It is expected that many diseases resulting from thedysfunction of cells may be amenable to treatment by the administrationof human embryonic stem cells or human embryonic stem cell-derivedcells. The ability of pluripotent embryonic stem cells to differentiateand give rise to a plurality of specialized mature cells reveals thepotential application of these cells as a means to replace, restore, orcomplement damaged or diseased cells, tissues, and organs. However,scientific and ethical considerations have slowed the progress ofresearch using embryonic stem cells recovered from aborted embryos orembryos formed using in vitro fertilization techniques.

Adult stem cells are present only at low frequencies and exhibitrestricted differentiation potential and poor growth. A further problemassociated with using adult stems cells is that these cells are notimmunologically privileged, or can lose their immunological privilegeafter transplant, wherein the term “immunologically privileged” is usedto denote a state where the recipient's immune system does not recognizethe cells as foreign. Thus, only autologous transplants are possible inmost cases when adult stem cells are used. Most presently envisionedforms of stem cell therapy are essentially customized medical proceduresand therefore economic factors associated with such procedures limittheir wide ranging potential.

The restoration of expression of at least some measuredembryonic-specific genes has been observed in somatic cells followingfusion with embryonic stem cells. However, the resulting cells arehybrids, often with a tetraploid genotype, and therefore not suited asnormal or histocompatible cells for transplant purposes.

The use of somatic cell nuclear transfer has been shown to adequatelyreprogram somatic cell nuclear content to adopt pluripotency, however,raises a set of concerns beyond the moral status. The stresses placed onboth the egg cell and the introduced nucleus are enormous, leading to ahigh loss in resulting cells. Furthermore, the procedure has to beperformed manually under a microscope, and therefore, somatic cellnuclear transfer is very resource intensive. In addition, not all of thedonor cell's genetic information is transferred, as the donor cell'smitochondria that contain their own mitochondrial DNA are left behind.The resulting hybrid cells retain those mitochondrial structures whichoriginally belonged to the egg. As a consequence, clones are not perfectcopies of the donor of the nucleus.

A major step towards patient derived pluripotent cells was achieved byTakahashi et al. in 2006. It was shown that the overexpression ofdefined transcription factors (TFs) which are known to regulate andmaintain stem cell pluripotency (Takahasi et al., 2006, Cell 126,663-676; Schulz & Hoffmann, 2007, Epigenetics 2, 37-42) can induce apluripotent state of murine somatic fibroblasts, termed inducedpluripotent stem (iPS) cells. In this study the authors identifiedOCT3/4, SOX2, KLF4 and c-MYC as being required for iPS cell generation(Takahasi et al., 2006). In a subsequent study the authors showed thatthe same TFs are able to reprogram adult human fibroblasts (Takahasi etal., 2007, Cell 131, 861-872), while others attributed this activity toa modified TF-cocktail composed of OCT3/4, SOX2, NANOG and LIN28regarding human (Yu et al., 2007, Science Express) or murine fibroblasts(Wernig et al., 2007, Nature 448, 318-324). For those initial studies aswell as most subsequent studies the reprogramming TFs were overexpressedusing retro- or lentiviral vectors. Due to the silencing of viralpromoters these studies reproducibly show that the expression exogenousTFs is shut down during the reprogramming process (reviewed by Hotta &Ellis, 2008, J. Cell Biochem. 105, 940-948). Accordingly, thepluripotent state is maintained by activated endogenous transcriptionfactors. Furthermore, the silencing of the virally expressed TFs isprerequisite for the subsequent re-differentiation of iPS cells totissue specific precursors (Yu et al., 2007). A major disadvantage ofviral delivery is the stochastic reactivation of integrated retrovirusesencoding potent oncogenes, which in the case of c-MYC led to theinduction of tumors in chimeric mice (Okita et al., 2007, Nature 448,313-317). Meanwhile it has been demonstrated that the generation of iPScells is possible in absence of MYC (Nakagawa et al., 2008). Overall,only OCT4 and SOX2 have been reported being essential for thereprogramming, oncogenes like MYC and KLF4 seem to acts like enhancers(McDevitt & Palecek, 2008, Curr. Opin. Biotechnol. 19, 527-33).Accordingly it has been shown that other transforming gene products likeSV40 Large-T antigen or hTERT can improve the efficiency of iPSgeneration (Mali et al., 2008, Stem Cells 26, 1998-2005). As theepigenetic reprogramming involves chromatin remodelling the addition ofhistone deacetylase (HDAC) inhibitors (like valproic acid) or DNAmethyltransferase inhibitors (like 5′-azaC) greatly improve thereprogramming efficiency (Huangfu et al., 2008, Nat. Biotechnol. 26,795-797) and reduced the need for TFs to OCT4 and SOX2 (Huangfu et al.,2008, Nat. Biotechnol. 26, 1269-1275).

Another strategy to reduce the risk associated with retroviralintergration into the host genome is the use of non-integratingadenoviral vectors, which mediate a transient transgene expressionsufficient for reprogramming (Stadtfeld et al., 2008, Sciencexpress).Transgene integration is also avoided by the use of conventionaleukaryotic expression plasmids leading to transient gene expression. Sofar, with this strategy MEFs have been successfully reprogrammed to iPScells (Okita et al., 2008, Science 322, 949-53). Genomic integration hasnot been detected in this study, however, stable genomic integration ina small fraction of the cells of transfected plasmid DNA cannot becompletely excluded.

Adult human fibroblasts are easily derived from healthy donors or—infuture clinical applications—from patients without risky surgicalintervention. However, a recent study has shown that human keratinocytesare more easily and more efficiently reprogrammend to iPS cells, andthat e.g. hair follicle derived keratinocytes might be the better sourceof choice for patient derived iPS cells (Aasen et al., 2008, NatureBiotechnology).

There remains a need for improved technologies for reprogrammingdifferentiated somatic cells to produce reprogrammed cells suitable forresearch, testing for quality control, and for use in cell therapy inhigh number and with good quality.

The present invention provides technologies of producing reprogrammedcells avoiding the use of DNA. These technologies use cells that areeasily and inexpensively obtained in unlimited quantities and providereprogrammed cells useful in cell therapy. The approach according to thepresent invention completely lacks the risk of genomic integration andopens the possibility of reprogramming without modification of the hostgenome.

SUMMARY OF THE INVENTION

The present invention exploits the fact that, when provided withappropriate factors, a terminally differentiated cell's fate can beredirected to pluripotentiality. Specifically, the present inventionprovides technology for reprogramming an animal differentiated somaticcell to a cell having stem cell properties. This method allowsde-differentiation of one type of somatic cells into pluripotentstem-like cells using a defined system in vitro. The method of theinvention in one embodiment provides autologous (isogeneic) cell typesfor cell transplantation in the same individual that donated the initialsomatic cell sample.

According to the present invention, one or more somatic cells areprovided with RNA capable of expressing one or more factors that inducethe reprogramming of somatic cells to cells having stem cellcharacteristics. Expression of RNA capable of expressing these factorsconfers characteristics of an undifferentiated cell to a somatic celland facilitates reprogramming of the somatic cell.

Introduction of the factors in the form of RNA has the advantage,relative to the use of DNA constructs, that, for expression, RNA needonly get into the cytoplasm of the cells, not into the cell nucleus.Therefore RNA transfer is not dependent on the division activity of thecells to be transfected. Furthermore, the transfection rates attainablewith RNA are relatively high, for many cell types even >90%, andtherefore, there is no need for selection. The amounts of proteinachieved correspond to those in physiological expression.

Furthermore, according to the invention, it is possible to control theamount of RNA that is introduced into a cell as well as the stabilityand translation level of the RNA in the cell.

Hence the amount and time of expression of certain factors expressed bythe RNA in the cell can be adjusted as necessary. In this way it ispossible to simulate the effects of different levels of expression in acell and introduce RNA into a cell in amounts sufficient to inducereprogramming and de-differentiation of somatic cells to produce cellshaving stem cell characteristics, preferably in amounts sufficient toallow development of somatic cells into pluripotent cells.

Most importantly, transfected RNA does not result in significantintegration into the host genome. In contrast, transfection of DNA formedical use is considered as gene therapy. DNA transfer is associatedwith a significant risk of mutations in the host genome with theincreased risk of malignant transformation. Thus, RNA transfer has amuch better safety profile and is not regarded as gene therapy.Moreover, transfected RNA is degraded in the host cell within days. Thismeans that a stem cell induced by transfection of RNA is geneticallyidentical to an autologous natural stem cell. Thus, cell types andtissues obtained from such stem cells are genetically non-discriminablefrom their autologous natural counterparts. In contrast, a stem cellinduced by DNA transfection carries additional foreign genes. Alltissues which derive from such a recombinant stem cell carry the samegenetic markers and thus, exhibit an increased risk of malignanttransformation.

In one aspect, the present invention relates to a method for producingcells having stem cell characteristics comprising the steps of (i)providing a cell population comprising somatic cells, (ii) introducingRNA into at least a portion of said somatic cells said RNA whenintroduced into a somatic cell is capable of inducing the development ofstem cell characteristics, and (iii) allowing the development of cellshaving stem cell characteristics. In one embodiment, the RNA is derivedfrom an undifferentiated cell such as a stem cell, for example anembryonic stem cell or an adult stem cell. In this respect, the term“derived” denotes for the fact that the RNA either has been obtainedfrom the cell, e.g. by isolation and optionally fractionation, and thus,is an isolate of cellular RNA or a fraction thereof and/or has acomposition similar to the RNA composition of the cell from which it isderived, or a fraction thereof. In one embodiment, the RNA compriseswhole-cell RNA. In another embodiment, the RNA is specific for saidundifferentiated cell. In this embodiment, the RNA may be a fraction ofwhole-cell RNA. In one embodiment, the RNA has been obtained by in vitrotranscription.

Preferably, step (iii) comprises culturing the somatic cells underembryonic stem cell culture conditions, preferably conditions suitablefor maintaining pluripotent stem cells in an undifferentiated state.

According to the present invention, the RNA preferably is introducedinto said at least a portion of somatic cells by electroporation.

In one embodiment of the method of the invention, the stem cellcharacteristics comprise an embryonic stem cell morphology, wherein saidembryonic stem cell morphology preferably comprises morphologicalcriteria selected from the group consisting of compact colonies, highnucleus to cytoplasm ratio and prominent nucleoli. In certainembodiments, the cells having stem cell characteristics have normalkaryotypes, express telomerase activity, express cell surface markersthat are characteristic for embryonic stem cells and/or express genesthat are characteristic for embryonic stem cells. The cell surfacemarkers that are characteristic for embryonic stem cells may be selectedfrom the group consisting of stage-specific embryonic antigen-3(SSEA-3), SSEA-4, tumor-related antigen-1-60 (TRA-1-60), TRA-1-81, andTRA-2-49/6E and the genes that are characteristic for embryonic stemcells may be selected from the group consisting of endogenous OCT4,endogenous NANOG, growth and differentiation factor 3 (GDF3), reducedexpression 1 (REX1), fibroblast growth factor 4 (FGF4), embryoniccell-specific gene 1 (ESG1), developmental pluripotency-associated 2(DPPA2), DPPA4, and telomerase reverse transcriptase (TERT).

Preferably, the cells having stem cell characteristics arede-differentiated and/or reprogrammed somatic cells. Preferably, thecells having stem cell characteristics exhibit the essentialcharacteristics of embryonic stem cells such as a pluripotent state.Preferably, the cells having stem cell characteristics have thedevelopmental potential to differentiate into advanced derivatives ofall three primary germ layers. In one embodiment, the primary germ layeris endoderm and the advanced derivative is gut-like epithelial tissue.In a further embodiment, the primary germ layer is mesoderm and theadvanced derivative is striated muscle and/or cartilage. In an evenfurther embodiment, the primary germ layer is ectoderm and the advancedderivative is neural tissue and/or epidermal tissue. In one preferredembodiment, the cells having stem cell characteristics have thedevelopmental potential to differentiate into neuronal cells and/orcardiac cells.

In one embodiment, the somatic cells are embryonic stem cell derivedsomatic cells with a mesenchymal phenotype. In a preferred embodiment,the somatic cells are fibroblasts such as fetal fibroblasts or postnatalfibroblasts or keratinocytes, preferably hair follicle derivedkeratinocytes. In further embodiments, the fibroblasts are lungfibroblasts, foreskin fibroblasts or dermal fibroblasts. In particularembodiments, the fibroblasts are fibroblasts as deposited at theAmerican Type Culture Collection (ATCC) under Catalog No. CCL-186 or asdeposited at the American Type Culture Collection (ATCC) under CatalogNo. CRL-2097. In one embodiment, the fibroblasts are adult human dermalfibroblast. Preferably, the somatic cells are human cells. According tothe present invention, the somatic cells may be genetically modified.

In a further aspect, the present invention relates to a method forproducing cells having stem cell characteristics comprising the steps of(i) providing a cell population comprising somatic cells, (ii)introducing RNA capable of expressing OCT4 and RNA capable of expressingSOX2 into at least a portion of said somatic cells and (iii) allowingthe development of cells having stem cell characteristics. In oneembodiment, the method further comprises introducing RNA capable ofexpressing NANOG and/or RNA capable of expressing LIN28 and,alternatively or additionally, further comprises introducing RNA capableof expressing KLF4 and/or RNA capable of expressing c-MYC.

In one embodiment, step (ii) comprises introducing RNA capable ofexpressing OCT4, RNA capable of expressing SOX2, RNA capable ofexpressing NANOG and RNA capable of expressing LIN28 into at least aportion of said somatic cells.

In another embodiment, step (ii) comprises introducing RNA capable ofexpressing OCT4, RNA capable of expressing SOX2, RNA capable ofexpressing KLF4 and RNA capable of expressing c-MYC into at least aportion of said somatic cells.

Preferably, step (iii) comprises culturing the somatic cells underembryonic stem cell culture conditions, preferably conditions suitablefor maintaining pluripotent stem cells in an undifferentiated state.

According to the present invention, the RNA preferably is introducedinto said at least a portion of somatic cells by electroporation.

In one embodiment of the method of the invention, the stem cellcharacteristics comprise an embryonic stem cell morphology, wherein saidembryonic stem cell morphology preferably comprises morphologicalcriteria selected from the group consisting of compact colonies, highnucleus to cytoplasm ratio and prominent nucleoli. In certainembodiments, the cells having stem cell characteristics have normalkaryotypes, express telomerase activity, express cell surface markersthat are characteristic for embryonic stem cells and/or express genesthat are characteristic for embryonic stem cells. The cell surfacemarkers that are characteristic for embryonic stem cells may be selectedfrom the group consisting of stage-specific embryonic antigen-3(SSEA-3), SSEA-4, tumor-related antigen-1-60 (TRA-1-60), TRA-1-81, andTRA-2-49/6E and the genes that are characteristic for embryonic stemcells may be selected from the group consisting of endogenous OCT4,endogenous NANOG, growth and differentiation factor 3 (GDF3), reducedexpression 1 (REX1), fibroblast growth factor 4 (FGF4), embryoniccell-specific gene 1 (ESG1), developmental pluripotency-associated 2(DPPA2), DPPA4, and telomerase reverse transcriptase (TERT).

Preferably, the cells having stem cell characteristics arede-differentiated and/or reprogrammed somatic cells. Preferably, thecells having stem cell characteristics exhibit the essentialcharacteristics of embryonic stem cells such as a pluripotent state.Preferably, the cells having stem cell characteristics have thedevelopmental potential to differentiate into advanced derivatives ofall three primary germ layers. In one embodiment, the primary germ layeris endoderm and the advanced derivative is gut-like epithelial tissue.In a further embodiment, the primary germ layer is mesoderm and theadvanced derivative is striated muscle and/or cartilage. In an evenfurther embodiment, the primary germ layer is ectoderm and the advancedderivative is neural tissue and/or epidermal tissue. In one preferredembodiment, the cells having stem cell characteristics have thedevelopmental potential to differentiate into neuronal cells and/orcardiac cells.

In one embodiment, the somatic cells are embryonic stem cell derivedsomatic cells with a mesenchymal phenotype. In a preferred embodiment,the somatic cells are fibroblasts such as fetal fibroblasts or postnatalfibroblasts or keratinocytes, preferably hair follicle derivedkeratinocytes. In further embodiments, the fibroblasts are lungfibroblasts, foreskin fibroblasts or dermal fibroblasts. In particularembodiments, the fibroblasts are fibroblasts as deposited at theAmerican Type Culture Collection (ATCC) under Catalog No. CCL-186 or asdeposited at the American Type Culture Collection (ATCC) under CatalogNo. CRL-2097. In one embodiment, the fibroblasts are adult human dermalfibroblast. Preferably, the somatic cells are human cells. According tothe present invention, the somatic cells may be genetically modified.

In a further aspect, the present invention relates to a method forreprogramming an animal differentiated somatic cell to a cell havingstem cell properties, comprising the step of introducing RNA capable ofexpressing one or more factors allowing the reprogramming of saidsomatic cell to a cell having stem cell characteristics into saidsomatic cell.

In one embodiment, the RNA is derived from an undifferentiated cell suchas a stem cell, for example an embryonic stem cell or an adult stemcell. In one embodiment, the RNA comprises whole-cell RNA. In anotherembodiment, the RNA is specific for said undifferentiated cell. In thisembodiment, the RNA may be a fraction of whole-cell RNA. In oneembodiment, the RNA has been obtained by in vitro transcription. Indifferent embodiments, said one or more factors capable of beingexpressed by the RNA comprise an assembly of factors selected from thegroup consisting of (i) OCT4 and SOX2, (ii) OCT4, SOX2, and one or bothof NANOG and LIN28, (iii) OCT4, SOX2 and one or both of KLF4 and c-MYC.In one embodiment, said one or more factors capable of being expressedby the RNA comprise OCT4, SOX2, NANOG and LIN28 or OCT4, SOX2, KLF4 andc-MYC. Preferably, the RNA is introduced into said animal differentiatedsomatic cell by electroporation or microinjection. Preferably, themethod further comprises allowing the development of cells having stemcell characteristics, e.g. by culturing the somatic cell under embryonicstem cell culture conditions, preferably conditions suitable formaintaining pluripotent stem cells in an undifferentiated state.

In one embodiment of the method of the invention, the stem cellcharacteristics comprise an embryonic stem cell morphology, wherein saidembryonic stem cell morphology preferably comprises morphologicalcriteria selected from the group consisting of compact colonies, highnucleus to cytoplasm ratio and prominent nucleoli. In certainembodiments, the cell having stem cell characteristics has a normalkaryotype, expresses telomerase activity, expresses cell surface markersthat are characteristic for embryonic stem cells and/or expresses genesthat are characteristic for embryonic stem cells. The cell surfacemarkers that are characteristic for embryonic stem cells may be selectedfrom the group consisting of stage-specific embryonic antigen-3(SSEA-3), SSEA-4, tumor-related antigen-1-60 (TRA-1-60), TRA-1-81, andTRA-2-49/6E and the genes that are characteristic for embryonic stemcells may be selected from the group consisting of endogenous OCT4,endogenous NANOG, growth and differentiation factor 3 (GDF3), reducedexpression 1 (REX1), fibroblast growth factor 4 (FGF4), embryoniccell-specific gene 1 (ESG1), developmental pluripotency-associated 2(DPPA2), DPPA4, and telomerase reverse transcriptase (TERT).

Preferably, the cell having stem cell characteristics is ade-differentiated and/or reprogrammed somatic cell. Preferably, the cellhaving stem cell characteristics exhibits the essential characteristicsof embryonic stem cells such as a pluripotent state. Preferably, thecell having stem cell characteristics has the developmental potential todifferentiate into advanced derivatives of all three primary germlayers. In one embodiment, the primary germ layer is endoderm and theadvanced derivative is gut-like epithelial tissue. In a furtherembodiment, the primary germ layer is mesoderm and the advancedderivative is striated muscle and/or cartilage. In an even furtherembodiment, the primary germ layer is ectoderm and the advancedderivative is neural tissue and/or epidermal tissue. In one preferredembodiment, the cell having stem cell characteristics has thedevelopmental potential to differentiate into neuronal cells and/orcardiac cells.

In one embodiment, the animal differentiated somatic cell is anembryonic stem cell derived somatic cell with a mesenchymal phenotype.In a preferred embodiment, the somatic cell is a fibroblast such asfetal fibroblast or postnatal fibroblast or a keratinocyte, preferablyhair follicle derived keratinocyte. In further embodiments, thefibroblast is a lung fibroblast, foreskin fibroblast or dermalfibroblast. In particular embodiments, the fibroblast is a fibroblast asdeposited at the American Type Culture Collection (ATCC) under CatalogNo. CCL-186 or as deposited at the American Type Culture Collection(ATCC) under Catalog No. CRL-2097. In one embodiment, the fibroblast isan adult human dermal fibroblast. Preferably, the animal differentiatedsomatic cell is a human cell. According to the present invention, theanimal differentiated somatic cell may be genetically modified.

Particular embodiments of the methods of the present invention furthercomprise the step of cryopreserving the cells having stem cellcharacteristics.

In further aspects, the present invention relates to cells having stemcell characteristics prepared by the methods of the present inventionand a composition of cells having stem cell characteristics prepared bythe methods of the present invention. In one embodiment, the compositionis a pharmaceutical composition.

In further aspects, the present invention relates to the use of thecells or the composition of the present invention in medicine, inparticular in transplantation medicine, for producing a disease model orfor drug development.

In a further aspect, the present invention relates to a method ofderiving differentiated cell types comprising the step of culturing thecells having stem cell characteristics of the present invention or thecomposition of cells having stem cell characteristics of the presentinvention under conditions that induce or direct partial or completedifferentiation to a particular cell type. In one embodiment, theconditions that induce or direct partial or complete differentiation toa particular cell type comprise the presence of at least onedifferentiation factor. Preferably, the somatic cell type of thedifferentiated cells obtained according to the present invention isdifferent from the somatic cell type of the somatic cells used forde-differentiation. Preferably, the de-differentiated cells are derivedfrom fibroblastic cells and said re-differentiated cell types aredifferent from fibroblastic cells. In another embodiment, thede-differentiated cells are derived from keratinocytes and saidre-differentiated cell types are different from keratinocytes.

In a further aspect, the present invention relates to an assay toidentify one or more factors useful for reprogramming an animaldifferentiated somatic cell to a cell having stem cell characteristicscomprising the steps of introducing RNA capable of expressing one ormore factors into said somatic cell and determining whether said somaticcell has developed into a cell having stem cell characteristics.Preferably, the method further comprises the step of allowing thedevelopment of cells having stem cell characteristics, e.g. by culturingthe animal differentiated somatic cell under embryonic stem cell cultureconditions, preferably conditions suitable for maintaining pluripotentstem cells in an undifferentiated state. Preferably, the RNA isintroduced into said animal differentiated somatic cell byelectroporation or microinjection.

In one embodiment, the step of determining whether said somatic cell hasdeveloped into a cell having stem cell characteristics comprisescomparing the gene expression of the cell obtained by the method of thepresent invention with gene expression found in embryonic stem cells,preferably of the same cell type, to determine whether said one or morefactors play a role in cellular reprogramming.

In one embodiment, the step of introducing RNA capable of expressing oneor more factors into said somatic cell comprises introducing RNA capableof expressing factors known to be involved in reprogramming an animaldifferentiated somatic cell to a cell having stem cell characteristics.In one embodiment, said factors known to be involved in reprogramming ananimal differentiated somatic cell to a cell having stem cellcharacteristics include at least one factor selected from the groupconsisting of OCT4, SOX2, NANOG, LIN28, KLF4 and c-MYC. In oneembodiment, said factors known to be involved in reprogramming an animaldifferentiated somatic cell to a cell having stem cell characteristicsinclude a combination of OCT4 and SOX2, a combination of OCT4, SOX2,NANOG and/or LIN28 and a combination of OCT4, SOX2, KLF4 and/or c-MYC.

Various embodiments of the somatic cell, the cell having stem cellcharacteristics and the culture conditions for allowing the developmentof cells having stem cell characteristics are as described above for themethods according to the other aspects of the present invention.

In a further aspect, the present invention relates to a kit forproducing cells having stem cell characteristics comprising RNA capableof expressing one or more factors known to be involved in reprogrammingan animal differentiated somatic cell to a cell having stem cellcharacteristics. Preferably, said kit comprises RNA capable ofexpressing OCT4 and RNA capable of expressing SOX2 and preferablyfurther comprises (i) RNA capable of expressing NANOG and/or RNA capableof expressing LIN28 and/or (ii) RNA capable of expressing KLF4 and/orRNA capable of expressing c-MYC. The kit may further comprise anembryonic stem cell culture medium.

In even a further aspect, the present invention relates to apharmaceutical composition comprising RNA which when introduced into asomatic cell is capable of inducing the development of stem cellcharacteristics in said cell. Particular embodiments of the RNA, thesomatic cell and the cell having stem cell characteristics are asdescribed herein for the other aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Determination of the influence of time on the amount oftranscript and protein following transfection of 786-0 cells with 20 μgeGFP IVT (in vitro transcribed) RNA and 2dGFP IVT RNA, respectively.Determination of the average fluorescence intensity of eGFP usingFACS-Kalibur.

FIGS. 2A and 2B: Scatter blot of all genes for determining thesimilarity of the duplicates.

FIG. 2A: Comparison of the duplicates of cells transfected with SYT-SSX2or eGFP;

FIG. 2B: Representation of replicates after 24 h.

FIGS. 3A and 3B: Representation of the genes regulated by SYT-SSX2.

FIG. 3A: Scatter blot of all analyzed genes. Comparison ofSYT-SSX2-transfected cells with eGFP-transfected cells followingtransfection of each 15 μg IVT RNA. Representation of replicates after24 h. Yellow-orange shows differential expression in the non-significantregion, i.e. below a factor of two, red and green show up- anddownregulation, respectively, by a factor greater than two.

FIG. 3B: Representation of the number of genes which are significantlyregulated after 8 h and 24 h, respectively, by transfection of 15 μg IVTRNA of the respective gene.

FIGS. 4A1-4A3 and 4B: Overview of different 5′-CAP-structures.

FIGS. 4A1-4A3: Pictured are the natural 5′CAP-structure of mRNA andchemical modified versions of this 5′-CAP (ARCA, D1 and D2-D1 and D2refer to the two diastereoisomers produced by the phosphorothioatemoiety), which were shown to stabilize mRNA.

FIG. 4B: Schematic overview of in vitro translated mRNA (IVT-RNA)synthesis.

FIG. 5: Electroporation of human fibroblasts (CCD1079Sk) and mouseembryonic fibroblasts (MEFs).

CCD1079Sk fibroblasts and MEFs were electroporated once with 10 μgIVT-RNA encoding eGFP. Voltage and capacity were chosen as indicated. 24h post electroporation the transfection efficiencies [%] were measuredby FACS, mean fluorescence levels are given in parenthesis.

FIG. 6: IVT RNA cap structure optimization.

CCD1079Sk fibroblasts were electroporated (250V, 300 μF) either withIVT-RNA of ARCA-luc (encoding Luciferase (luc) with ARCA-5′-CAP), D1-lucor D2-luc (each 10 μg). After 2 h, 4 h, 8 h, 24 h, 48 h, and 72 hluciferase assays were performed in duplicates. Data are expressed asmean luciferase activity ±SD.

FIG. 7: Persistence of electroporated IVT-RNA in human fibroblasts.

CCD1079Sk fibroblasts were electroporated once with 15 μg IVT-RNA ofeach transcription factor. The intracellular levels of these IVT-RNAconstructs were quantified by qRT-PCR 7 days post electroporation.

FIGS. 8A and 8B: Expression of human and murine transcription factorsafter electroporation of IVT-RNA constructs.

MEFs (FIG. 8A) and CCD1079Sk fibroblasts (FIG. 8B) were electroporatedonce with respectively 10 μg or 2,5 μg IVT-RNA encoding the fourtranscription factors (TFs) OCT4, SOX2, KLF4 and c-MYC (OSKM). Cellswere lysed at the indicated timepoints post electroporation. The proteinexpression was monitored by Western Bloting using specific antibodies.293T-cells electroporated with μg IVT-RNA encoding OSKM were used aspositive control.

FIGS. 9A and 9B: Alkaline phosphatase staining of electroporated humanCCD1079Sk fibroblasts.

FIG. 9A: CCD1079Sk fibroblasts were electroporated once either withIVT-RNA encoding the four TFs OSKM or with buffer (mock) and cultivatedin human ES cell medium.

FIG. 9B: After 10 days cells were stained for alkaline phosphatase (AP)and the resulting red flourescence was monitored by FACS.

FIGS. 10A and 10B: Alkaline phosphatase staining of electroporated humanCCD1079Sk fibroblasts.

FIG. 10A: CCD1079Sk cells were electroporated three consecutive times in48 h intervals with IVT-RNA encoding either GFP (mock) or the four TFsOSKM (2.5 or 1.25 μg each). OSKM or mock transfected cells werecultivated in iPS medium.

FIG. 10B: After 192 h cells were stained for alkaline phosphatase (AP)and monitored by fluorescence microscopy.

FIGS. 11A and 11B: Alkaline phosphatase staining of electroporated MEFs.

FIG. 11A: MEFs cultivated until passage 3 were electroporated in 48 hintervals with IVT-RNA encoding either GFP (mock) or the four murine TFsOSKM (5 μg each). OSKM or mock transfected MEFs were cultivated in mouseES cell medium in the presence or absence of 2 mM valproic acid (VPA) asindicated.

FIG. 11B: After 96 h cells were stained for alkaline phosphatase (AP)and monitored by fluorescence microscopy.

FIGS. 12A and 12B: Expression of human ES-marker genes of electroporatedCCD1079Sk cells.

FIG. 12A: CCD1079Sk fibroblasts were electroporated two times eitherwith buffer (mock) or with 15 μg IVT-RNA encoding the transcriptionfactors OSKM and cultivated in human ES cell medium in the presence orabsence of VPA (0.5 or 1 mM) as indicated.

FIG. 12B: After the indicated time points, 10% of the cells were removedfrom the cultures prior to subsequent electroporation, total RNA wasisolated and mRNA-expression of the human ES-marker genes OCT4(endogenous), TERT and GDF3 was evaluated by real-time PCR.

FIGS. 13A and 13B: Expression of human ES-marker genes of electroporatedCCD1079Sk cells.

FIG. 13A: CCD1079Sk fibroblasts were electroporated as indicated eitherwith 15 μg or 5 μg IVT-RNA encoding the transcription factors OSKM orwith buffer (mock) and cultivated in human ES cell medium in thepresence or absence of 1 mM VPA as indicated.

FIG. 13B: After the indicated time points, 10% of the cells were removedfrom the cultures prior to subsequent electroporation, total RNA wasisolated and mRNA-expression of the human ES-marker genes OCT4(endogenous), TERT, GDF3 and DPPA4 was quantified by qRT-PCR.

FIGS. 14A and 14B: Expression of human ES-marker genes of electroporatedCCD1079Sk cells.

FIG. 14A: MEFs were electroporated six consecutive times with 5 or 2.5μg IVT-RNA encoding either GFP (mock) or the four murine transcriptionfactors OSKM and cultivated in mouse ES cell medium in the presence orabsence of 2 mM VPA as indicated.

FIG. 14B: After the indicated time points, 10% of the cells were removedfrom the cultures prior to subsequent electroporation, total RNA wasisolated and mRNA-expression of the murine ES-marker gene mTert wasevaluated by qRT-PCR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides technology to change one type of highlyspecialized somatic cells, e.g. fibroblasts or keratinocytes, intoanother type, e.g., neuronal cells, via a pluripotent cell intermediate.

Specifically, by providing a differentiated somatic cell with factorspresent in pluripotent cell types, preferably stem cells, morepreferably embryonic stem cells, the invention restores the cell'sepigenetic memory to a state similar to that of pluripotent stem cells.With the present invention, embryos do not have to be used, created, ordestroyed to generate cells having stem cell characteristics, inparticular pluripotency, thus eliminating ethical concerns. Furthermore,the present invention does not require the use of vectors that integrateinto the genome such as viral vectors potentially introducing mutationsat the insertion site.

The somatic cells used according to the present invention have animportant advantage over oocytes as a means of inducing reprogramming inthat they can be easily expanded in number in vitro. In addition, thepresent invention allows the use of patient-specific somatic cells andthus, largely eliminates the concerns of immune rejection and problemsassociated with patient immunosuppression. Using cells generatedaccording to the present invention for autologous cell transplantationis unlikely to induce adverse side effects and/or resistance. Ifrequired, repeated cell transplantation is feasible. However, since thepresent invention will significantly reduce the need forimmunosuppression of the patient to reduce acute and hyperacuterejection the need for repeated transplantation procedures will also bealleviated, reducing the cost of disease treatment.

Terms such as “cell having stem cell characteristics”, “cell having stemcell properties” or “stem like cell” are used herein to designate cellswhich, although they are derived from differentiated somatic non-stemcells, exhibit one or more features typical for stem cells, inparticular embryonic stem cells. Such features include an embryonic stemcell morphology such as compact colonies, high nucleus to cytoplasmratio and prominent nucleoli, normal karyotypes, expression oftelomerase activity, expression of cell surface markers that arecharacteristic for embryonic stem cells, and/or expression of genes thatare characteristic for embryonic stem cells. The cell surface markersthat are characteristic for embryonic stem cells are, for example,selected from the group consisting of stage-specific embryonic antigen-3(SSEA-3), SSEA-4, tumor-related antigen-1-60 (TRA-1-60), TRA-1-81, andTRA-2-49/6E. The genes that are characteristic for embryonic stem cellsare selected, for example, from the group consisting of endogenous OCT4,endogenous NANOG, growth and differentiation factor 3 (GDF3), reducedexpression 1 (REX1), fibroblast growth factor 4 (FGF4), embryoniccell-specific gene 1 (ESG1), developmental pluripotency-associated 2(DPPA2), DPPA4, and telomerase reverse transcriptase (TERT). In oneembodiment, the one or more features typical for stem cells includepluripotency.

A “stem cell” is a cell with the ability to self-renew, to remainundifferentiated, and to become differentiated. A stem cell can dividewithout limit, for at least the lifetime of the animal in which itnaturally resides. A stem cell is not terminally differentiated; it isnot at the end stage of a differentiation pathway. When a stem celldivides, each daughter cell can either remain a stem cell or embark on acourse that leads toward terminal differentiation.

Totipotent stem cells are cells having totipotential differentiationproperties and being capable of developing into a complete organism.This property is possessed by cells up to the 8-cell stage afterfertilization of the oocyte by the sperm. When these cells are isolatedand transplanted into the uterus, they can develop into a completeorganism.

Pluripotent stem cells are cells capable of developing into variouscells and tissues derived from the ectodermal, mesodermal and endodermallayers. Pluripotent stem cells which are derived from the inner cellmass located inside of blastocysts, generated 4-5 days afterfertilization are called “embryonic stem cells” and can differentiateinto various other tissue cells but cannot form new living organisms.

Multipotent stem cells are stem cells differentiating normally into onlycell types specific to their tissue and organ of origin. Multipotentstem cells are involved not only in the growth and development ofvarious tissues and organs during the fetal, neonatal and adult periodsbut also in the maintenance of adult tissue homeostasis and the functionof inducing regeneration upon tissue damage. Tissue-specific multipotentcells are collectively called “adult stem cells”.

An “embryonic stem cell” is a stem cell that is present in or isolatedfrom an embryo. It can be pluripotent, having the capacity todifferentiate into each and every cell present in the organism, ormultipotent, with the ability to differentiate into more than one celltype.

As used herein, “embryo” refers to an animal in the early stages of itdevelopment. These stages are characterized by implantation andgastrulation, where the three germ layers are defined and establishedand by differentiation of the germs layers into the respective organsand organ systems. The three germ layers are the endoderm, ectoderm andmesoderm.

A “blastocyst” is an embryo at an early stage of development in whichthe fertilized ovum has undergone cleavage, and a spherical layer ofcells surrounding a fluid-filled cavity is forming, or has formed. Thisspherical layer of cells is the trophectoderm. Inside the trophectodermis a cluster of cells termed the inner cell mass (ICM). Thetrophectoderm is the precursor of the placenta, and the ICM is theprecursor of the embryo.

An adult stem cell, also called a somatic stem cell, is a stem cellfound in an adult. An adult stem cell is found in a differentiatedtissue, can renew itself, and can differentiate, with some limitations,to yield specialized cell types of its tissue of origin. Examplesinclude mesenchymal stem cells, hematopoietic stem cells, and neuralstem cells.

A “differentiated cell” is a mature cell that has undergone progressivedevelopmental changes to a more specialized form or function. Celldifferentiation is the process a cell undergoes as it matures to anovertly specialized cell type. Differentiated cells have distinctcharacteristics, perform specific functions, and are less likely todivide than their less differentiated counterparts.

An “undifferentiated” cell, for example, an immature, embryonic, orprimitive cell, typically has a nonspecific appearance, may performmultiple, non-specific activities, and may perform poorly, if at all, infunctions typically performed by differentiated cells.

The term “autologous” is used to describe anything that is derived froman organism's own tissues, cells, or DNA. For example, “autologoustransplant” refers to a transplant of tissue or organs derived from thesame organism. Such procedures are advantageous because they overcomethe immunological barrier which otherwise results in rejection.

The term “heterologous” is used to describe something consisting ofmultiple different elements. As an example, the transfer of oneindividual's bone marrow into a different individual constitutes aheterologous transplant. A heterologous gene is a gene derived from asource other than the organism.

“Somatic cell” refers to any and all differentiated cells and does notinclude stem cells, germ cells, or gametes. Preferably, “somatic cell”as used herein refers to a terminally differentiated cell.

As used herein, “committed” refers to cells which are considered to bepermanently committed to a specific function. Committed cells are alsoreferred to as “terminally differentiated cells”.

As used herein, “differentiation” refers to the adaptation of cells fora particular form or function. In cells, differentiation leads to a morecommitted cell.

As used herein, “de-differentiation” refers to loss of specialization inform or function. In cells, de-differentiation leads to a less committedcell.

As used herein “reprogramming” refers to the resetting of the geneticprogram of a cell. A reprogrammed cell preferably exhibits pluripotency.

The terms “de-differentiated” and “reprogrammed” or similar terms areused interchangeably herein to denote somatic cell-derived cells havingstem cell characteristics. However, said terms are not intended to limitthe subject-matter disclosed herein by mechanistic or functionalconsiderations.

The term “RNA inducing the development of stem cell characteristics”refers to RNA which when introduced into a somatic cell induces the cellto de-differentiate.

As used herein, “germ cell” refers to a reproductive cell such as aspermatocyte or an oocyte, or a cell that will develop into areproductive cell.

As used herein, “pluripotent” refers to cells that can give rise to anycell type except the cells of the placenta or other supporting cells ofthe uterus.

According to the invention, standard methods can be used for productionof nucleic acids, cultivation of cells and introduction of RNA intocells.

According to the invention, the term “nucleic acid” comprisesdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinationsthereof, and modified forms thereof. The term comprises genomic DNA,cDNA, mRNA, recombinantly produced and chemically synthesized molecules.According to the invention, a nucleic acid may be present as asingle-stranded or double-stranded and linear or covalently circularlyclosed molecule.

A nucleic acid can, according to the invention, be isolated. The term“isolated nucleic acid” means, according to the invention, that thenucleic acid (i) was amplified in vitro, for example by a polymerasechain reaction (PCR), (ii) was produced recombinantly by cloning, (iii)was purified, for example by cleavage and separation by gelelectrophoresis or (iv) was synthesized, for example by chemicalsynthesis.

In a preferred embodiment, a cloned nucleic acid is, according to theinvention, present in a vector, with the vector optionally comprising apromoter that controls the expression of the nucleic acid. The term“vector” is used in its most general meaning and comprises anyintermediate vehicles for a nucleic acid that make it possible, forexample, to insert the nucleic acid into prokaryotic and/or eukaryoticcells and optionally integrate it into a genome.

Such vectors are preferably replicated and/or expressed in the cell. Anintermediate vehicle can be adapted e.g. for use in electroporation, inmicroprojectile bombardment, in liposomal administration, in transfer bymeans of agrobacteria or in insertion via DNA or RNA viruses. Vectorscomprise plasmids, phagemids or viral genomes.

The term “gene” relates according to the invention to a particularnucleic acid sequence, which is responsible for the production of one ormore cellular products and/or for the attainment of one or moreintercellular or intracellular functions. In particular the term relatesto a DNA segment that codes for a specific protein or a functional orstructural RNA molecule.

As used herein, the term “RNA” means a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′-position of a beta-D-ribo-furanose moiety. Theterm includes double stranded RNA, single stranded RNA, isolated RNAsuch as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of a RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in RNA molecules can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

According to the present invention, the term “RNA” includes andpreferably relates to “mRNA” which means “messenger RNA” and relates toa “transcript” which may be produced using DNA as template and encodes apeptide or protein. mRNA typically comprises a 5′ non translated region,a protein or peptide coding region and a 3′ non translated region. mRNAhas a limited halftime in cells and in vitro. Preferably, mRNA isproduced by in vitro transcription using a DNA template.

The term “expression” is used according to the invention in its mostgeneral meaning and comprises the production of RNA or of RNA andproteins/peptides, e.g. by transcription and/or translation. It alsocomprises partial expression of nucleic acids. Moreover, expression canbe transient or stable. With reference to RNA, the term “expression”relates in particular to the production of proteins/peptides.

Expression control sequences or regulatory sequences, which according tothe invention may be linked functionally with a nucleic acid, can behomologous or heterologous with respect to the nucleic acid. A codingsequence and a regulatory sequence are linked together “functionally” ifthey are bound together covalently, so that the transcription ortranslation of the coding sequence is under the control or under theinfluence of the regulatory sequence. If the coding sequence is to betranslated into a functional protein, with functional linkage of aregulatory sequence with the coding sequence, induction of theregulatory sequence leads to a transcription of the coding sequence,without causing a reading frame shift in the coding sequence orinability of the coding sequence to be translated into the desiredprotein or peptide.

The term “expression control sequence” or “regulatory sequence”comprises, according to the invention, promoters, ribosome-bindingsequences and other control elements, which control the transcription ofthe gene or the translation of the derived RNA. In certain embodimentsof the invention, the expression control sequences can be controlled.The precise structure of regulatory sequences can vary depending on thespecies or depending on the cell type, but generally comprises5′-untranscribed and 5′- and 3′-untranslated sequences, which areinvolved in the initiation of transcription or translation, such asTATA-box, capping-sequence, CAAT-sequence and the like. In particular,5′-untranscribed regulatory sequences comprise a promoter region thatincludes a promoter sequence for transcriptional control of thefunctionally bound gene. Regulatory sequences can also comprise enhancersequences or upstream activator sequences.

The term “transcription” according to the invention relates to theprocess by which the genetic code in a DNA sequence is transcribed intoRNA. The RNA may subsequently be translated into protein. According tothe invention, the term “transcription” comprises “invitro-transcription” (IVT) which relates to a process, wherein RNA, inparticular mRNA, is synthesized in a cell free system in vitropreferably using appropriately prepared cell extracts. Preferablycloning vectors are used for producing transcripts which generally aredesignated transcription vectors.

The term “translation” according to the invention relates to the processin the ribosomes of a cell by which a strand of messenger RNA directsthe assembly of a sequence of amino acids to make a protein or peptide.

In particular embodiments, the RNA that is to be introduced into a cellaccording to the invention comprises a population of different RNAmolecules, e.g. whole-cell RNA, an RNA library, or a portion of thereof,e.g. a library of RNA molecules expressed in a particular cell type,such as undifferentiated cells, in particular stem cells such asembryonic stem cells, or a fraction of the library of RNA molecules suchas RNA with enriched expression in undifferentiated cells, in particularstem cells such as embryonic stem cells relative to differentiatedcells.

Thus, according to the invention, the term “RNA” may include whole-cellRNA or a fraction thereof, which may be obtained by a process comprisingthe isolation of RNA from cells and/or by recombinant means, inparticular by in vitro transcription.

In one embodiment of the methods according to the invention, the RNAthat is to be introduced into a cell is obtained by in vitrotranscription of an appropriate DNA template. The promoter forcontrolling transcription can be any promoter for an RNA polymerase.Particular examples of RNA polymerases are the T7, T3 and SP6 RNApolymerases. Preferably the in vitro transcription according to theinvention is controlled by a T7 or SP6 promoter.

A DNA template for in vitro transcription may be obtained by cloning ofa nucleic acid, in particular cDNA, and introducing it into anappropriate vector for in vitro transcription. The cDNA may be obtainedby reverse transcription of RNA. The cDNA containing vector template maycomprise vectors carrying different cDNA inserts which followingtranscription results in a population of different RNA moleculesoptionally capable of expressing different factors or may comprisevectors carrying only one species of cDNA insert which followingtranscription only results in a population of one RNA species capable ofexpressing only one factor. Thus, it is possible to produce RNA capableof expressing a single factor only or to produce compositions ofdifferent RNAs such as RNA libraries and whole-cell RNA capable ofexpressing more than one factor, e.g. a composition of factors specificfor embryonic stem cells. The present invention envisions theintroduction of all such RNA into somatic cells.

In particular, for obtaining whole-cell RNA or a fraction thereof by invitro transcription one can proceed as follows: 1. RNA is isolated fromcells and the RNA is optionally fractionated to select a specificsubspecies of RNA for further processing. 2. The RNA thus obtained istransformed into cDNA, in particular by reverse transcription. 3. ThecDNA following an optional separation step to select a specificsubspecies of cDNA for further processing is inserted into a vectorsuitable for in vitro transcription. 4. The vector containing the cDNA(optionally following linearization of the vector) is subjected to invitro transcription. The optional step of fractionating RNA may serve toseparate RNA containing a poly-A sequence from RNA not containing suchsequence. Furthermore, it may serve to separate RNA according to forexample size, particular patterns of expression etc. For example, ifundifferentiated cells, in particular stem cells such as embryonic stemcells are used for isolating the RNA it is possible to select RNA forfurther processing which is specifically expressed in said cells butnot, for example, in differentiated cells. A similar fractionation ofcDNA is possible in step 3.

The RNA used according to the present invention may have a knowncomposition (in this embodiment it is preferably known which factors arebeing expressed by the RNA) or the composition of the RNA may bepartially or entirely unknown. Alternatively, the RNA used according tothe present invention may have a known function or the function of theRNA may be partially or entirely unknown.

The present invention also relates to a method for screening factorswhich, on introduction into a somatic cell, either alone or incombination with other factors, are capable of inducing, enhancing orinhibiting reprogramming of an animal differentiated somatic cell to acell having stem cell characteristics such as pluripotency. This methodcan also comprise determination of the nucleotide sequence of the RNAthat causes the observed effect on the animal differentiated somaticcell.

According to the invention, the term “RNA capable of expressing” withrespect to a particular factor means that the RNA, if present in theappropriate environment, preferably within a cell, can be expressed toproduce said factor. Preferably, RNA according to the invention is ableto interact with the cellular translation machinery to provide thefactor it is capable of expressing.

RNA capable of expressing a particular factor according to the presentinvention includes naturally occurring RNA capable of expressing saidfactor and any non-naturally occurring RNA capable of expressing saidfactor, e.g. modified forms or variants of naturally occurring RNAcapable of expressing said factor. For example, due to the degeneracy ofthe genetic code, the sequence of RNA can be modified without alteringthe sequence of the expressed factor. Furthermore, RNA may be modifiedto alter its stability and expression level.

The term “RNA capable of expressing” with respect to a particular factorincludes compositions only containing RNA encoding the factor andcompositions comprising RNA encoding the factor but also other RNA, inparticular RNA encoding different proteins/peptides. Thus, the term “RNAcapable of expressing” with respect to a particular factor may alsoinclude whole-cell RNA or a fraction thereof.

If according to the invention reference is made to RNA expressing morethan one factor, the RNA may comprise different RNA molecules expressingdifferent of these more than one factors. However, the present inventionalso includes situations wherein one RNA molecule expresses differentfactors, optionally linked through each other.

According to the invention, the stability and translation efficiency ofthe RNA introduced into a cell may be modified as required. For example,RNA may be stabilized and its translation increased by one or moremodifications having a stabilizing effects and/or increasing translationefficiency of RNA. Such modifications are described, for example, inPCT/EP2006/009448 incorporated herein by reference.

For example, RNA having an unmasked poly-A sequence is translated moreefficiently than RNA having a masked poly-A sequence. The term “poly-Asequence” relates to a sequence of adenyl (A) residues which typicallyis located on the 3′-end of a RNA molecule and “unmasked poly-Asequence” means that the poly-A sequence at the 3′ end of an RNAmolecule ends with an A of the poly-A sequence and is not followed bynucleotides other than A located at the 3′ end, i.e. downstream, of thepoly-A sequence. Furthermore, a long poly-A sequence of about 120 basepairs results in an optimal transcript stability and translationefficiency of RNA.

Therefore, in order to increase stability and/or expression of the RNAused according to the present invention, it may be modified so as to bepresent in conjunction with a poly-A sequence, preferably having alength of 10 to 500, more preferably 30 to 300, even more preferably 65to 200 and especially 100 to 150 adenosine residues. In an especiallypreferred embodiment the poly-A sequence has a length of approximately120 adenosine residues. To further increase stability and/or expressionof the RNA used according to the invention, the poly-A sequence can beunmasked.

In addition, incorporation of a 3′-non translated region (UTR) into the3′-non translated region of an RNA molecule can result in an enhancementin translation efficiency. A synergistic effect may be achieved byincorporating two or more of such 3′-non translated regions. The 3′-nontranslated regions may be autologous or heterologous to the RNA intowhich they are introduced. In one particular embodiment the 3′-nontranslated region is derived from the human β-globin gene.

A combination of the above described modifications, i.e. incorporationof a poly-A sequence, unmasking of a poly-A sequence and incorporationof one or more 3′-non translated regions, has a synergistic influence onthe stability of RNA and increase in translation efficiency.

In order to increase expression of the RNA used according to the presentinvention, it may be modified within the coding region, i.e. thesequence encoding the expressed factor, preferably without altering thesequence of the expressed factor, so as to increase the GC-content andthus, enhance translation in cells.

In further embodiments of the invention, the RNA that is to beintroduced into a cell has, at its 5′ end, a Cap structure or aregulatory sequence, which promotes the translation in the host cell.Preferably, RNA is capped at its 5′ end by an optionally modified7-methylguanosine attached by a 5′-5′ bridge to the first transcribednucleotide of the mRNA chain. Preferably, the 5′ end of the RNA includesa Cap structure having the following general formula:

wherein R₁ and R₂ are independently hydroxy or methoxy and W⁻, X⁻ and Y⁻are independently oxygen or sulfur. In a preferred embodiment, R₃ and 16are hydroxy and W, X⁻ and Y⁻ are oxygen. In a further preferredembodiment, one of R₁ and R₂, preferably R₁ is hydroxy and the other ismethoxy and W⁻, X⁻ and Y⁻ are oxygen. In a further preferred embodiment,R₁ and R₂ are hydroxy and one of W⁻, X⁻ and Y⁻, preferably X⁻ is sulfurwhile the other are oxygen. In a further preferred embodiment, one of R₁and R₂, preferably R₂ is hydroxy and the other is methoxy and one of W⁻,X⁻ and Y⁻, preferably X is sulfur while the other are oxygen.

In the above formula, the nucleotide on the right hand side is connectedto the RNA chain through its 3′ group. Preferred embodiments of the 5′Cap structure are also shown in FIG. 4A.

Those Cap structures wherein at least one of W⁻, X⁻ and Y⁻ is sulfur,i.e. which have a phosphorothioate moiety, exist in differentdiastereoisomefic forms all of which are encompassed herein.Furthermore, the present invention encompasses all tautomers andstereoisomers of the above formula.

For example, the Cap structure having the above structure wherein R₁ ismethoxy, R₂ is hydroxy, X is sulfur and W⁻ and Y⁻ are oxygen exists intwo diastereoisomeric forms (Rp and Sp). These can be resolved byreverse phase HPLC and are named D1 and D2 according to their elutionorder from the reverse phase HPLC column. According to the invention,the D1 isomer of m₂ ^(7,2′-o)Gpp_(S)pG is particularly preferred.

Of course, if according to the present invention it is desired todecrease stability and/or translation efficiency of RNA, it is possibleto modify RNA so as to interfere with the function of elements asdescribed above increasing the stability and or translation efficiencyof RNA.

According to the present invention, any technique useful fortransferring RNA into cells may be used for introducing RNA into cells.Preferably, RNA is transfected into cells by standard techniques. Suchtechniques include electroporation, lipofection and microinjection. Inone particularly preferred embodiment of the present invention, RNA isintroduced into cells by electroporation.

Electroporation or electropermeabilization relates to a significantincrease in the electrical conductivity and permeability of the cellplasma membrane caused by an externally applied electrical field. It isusually used in molecular biology as a way of introducing some substanceinto a cell.

Electroporation is usually done with electroporators, appliances whichcreate an electro-magnetic field in the cell solution. The cellsuspension is pipetted into a glass or plastic cuvette which has twoaluminum electrodes on its sides.

For electroporation, typically a cell suspension of around 50microliters is used. Prior to electroporation it is mixed with thenucleic acid to be transformed. The mixture is pipetted into thecuvette, the voltage and capacitance is set and the cuvette insertedinto the electroporator. Preferably, liquid medium is added immediatelyafter electroporation (in the cuvette or in an eppendorf tube), and thetube is incubated at the cells' optimal temperature for an hour or moreto allow recovery of the cells and optionally expression of antibioticresistance.

Preferably according to the invention a voltage of 200 to 300 V,preferably 230 to 270 V, more preferably around 250 V and a capacitanceof 200 to 600 μF, preferably 250 to 500 μF, more preferably preferably300 to 500 μF is used for electroporation.

According to the invention it is preferred that introduction of RNAcapable of expressing certain factors as disclosed herein into somaticcells results in expression of said factors for a time period tocomplete the reprogramming process and in the development of cellshaving stem cell characteristics. Preferably, introduction of RNAcapable of expression certain factors as disclosed herein into somaticcells results in expression of said factors for an extended period oftime, preferably for at least 10 days, preferably for at least 11 daysand more preferably for at least 12 days. To achieve such long termexpression, RNA is preferably periodically introduced into the cellsmore than one time, preferably using electroporation. Preferably, RNA isintroduced into the cells at least twice, more preferably at least 3times, more preferably at least 4 times, even more preferably at least 5times up to preferably 6 times, more preferably up to 7 times or even upto 8, 9 or 10 times to ensure expression of one or more factors for anextended period of time. Preferably, the time periods elapsing betweenthe repeated introductions of the RNA are from 24 hours to 120 hours,preferably 48 hours to 96 hours. In one embodiment, time periodselapsing between the repeated introductions of the RNA are not longerthan 72 hours, preferably not longer than 48 hours or 36 hours. In oneembodiment, prior to the next electroporation, cells are allowed torecover from the previous electroporation. In this embodiment, the timeperiods elapsing between the repeated introductions of the RNA are atleast 72 hours, preferably at least 96 hours, more preferably at least120 hours. In any case, the conditions should be selected so that thefactors are expressed in the cells in amount and for periods of timewhich support the reprogramming process.

Preferably at least 1 μg, preferably at least 1.25 μg, more preferablyat least 1.5 μg and preferably up to 20 μg, more preferably up to 15 μg,more preferably up to 10 μg, more preferably up to 5 μg, preferably 1 to10 μg, even more preferably 1 to 5 μg, or 1 to 2.5 μg of RNA for eachfactor is used per electroporation.

Preferably, to allow the development of cells having stem cellcharacteristics, cells are cultivated in the presence of one or more DNAmethyltransferase inhibitors and/or one or more histone deacetylaseinhibitors. Preferred compounds are selected from the group consistingof 5′-azacytidine (5′-azaC), suberoylanilide hydroxamic acid (SAHA),dexamethasone, trichostatin A (TSA) and valproic acid (VPA). Preferably,cells are cultivated in the presence of valproic acid (VPA), preferablyin a concentration of between 0.5 and 10 mM, more preferably between 1and 5 mM, most preferably in a concentration of about 2 mM.

In a preferred embodiment of the present invention, RNA is introducedinto the somatic cells by repeated electroporations. Preferably, if aloss of viability of the cells occurs, previously not electroporatedcells are added as carrier cells. Preferably, previously notelectroporated cells are added prior to, during or after one or more ofthe 4^(th) and subsequent, preferably, the 5^(th) and subsequentelectroporations such as prior to, during or after the 4^(th) and 6thelectroporation. Preferably, previously not electroporated cells areadded prior to, during or after the 4^(th) or 5^(th) and each subsequentelectroporation.

Preferably, introduction of RNA capable of expressing one or morefactors into a cell causes expression of the one or more factors in thecell.

The term “transfection of RNA” relates according to the invention to theintroduction of one or more nucleic acids into a cell. According to thepresent invention, the cell can be an isolated cell or it can form partof an organ, a tissue and/or an organism.

The term “factor” according to the invention when used in conjunctionwith the expression thereof by RNA includes proteins and peptides aswell as derivatives and variants thereof. For example, the term “factor”comprises OCT4, SOX2, NANOG, LIN28, KLF4 and c-MYC.

The factors can be of any animal species; e.g., mammals and rodents.Examples of mammals include but are not limited to human and non-humanprimates. Primates include but are not limited to humans, chimpanzees,baboons, cynomolgus monkeys, and any other New or Old World monkeys.Rodents include but are not limited to mouse, rat, guinea pig, hamsterand gerbil.

OCT4 is a transcription factor of the eukaryotic POU transcriptionfactors and an indicator of pluripotency of embryonic stem cells. It isa maternally expressed Octomer binding protein. It has been observed tobe present in oocytes, the inner cell mass of blastocytes and also inthe primordial germ cell. The gene POU5F1 encodes the OCT4 protein.Synonyms to the gene name include OCT3, OCT4, OTF3 and MGC22487. Thepresence of OCT4 at specific concentrations is necessary for embryonicstem cells to remain undifferentiated.

Preferably, “OCT4 protein” or simply “OCT4” relates to human OCT4 andpreferably comprises an amino acid sequence encoded by the nucleic acidaccording to SEQ ID NO: 1, preferably the amino acid sequence accordingto SEQ ID NO: 2. One skilled in the art would understand that the cDNAsequence of OCT4 as described above would be equivalent to OCT4 mRNA,and can be used for the generation of RNA capable of expressing OCT4.

Sox2 is a member of the Sox (SRY-related HMG box) gene family thatencode transcription factors with a single HMG DNA-binding domain. SOX2has been found to control neural progenitor cells by inhibiting theirability to differentiate. The repression of the factor results indelamination from the ventricular zone, which is followed by an exitfrom the cell cycle. These cells also begin to lose their progenitorcharacter through the loss of progenitor and early neuronaldifferentiation markers.

Preferably, “SOX2 protein” or simply “SOX2” relates to human SOX2 andpreferably comprises an amino acid sequence encoded by the nucleic acidaccording to SEQ ID NO: 3, preferably the amino acid sequence accordingto SEQ ID NO: 4. One skilled in the art would understand that the cDNAsequence of SOX2 as described above would be equivalent to SOX2 mRNA,and can be used for the generation of RNA capable of expressing SOX2.

NANOG is a NK-2 type homeodomain gene, and has been proposed to play akey role in maintaining stem cell pluripotency presumably by regulatingthe expression of genes critical to embryonic stem cell renewal anddifferentiation. NANOG behaves as a transcription activator with twounusually strong activation domains embedded in its C terminus.Reduction of NANOG expression induces differentiation of embryonic stemcells.

Preferably, “NANOG protein” or simply “NANOG” relates to human NANOG andpreferably comprises an amino acid sequence encoded by the nucleic acidaccording to SEQ ID NO: 5, preferably the amino acid sequence accordingto SEQ ID NO: 6. One skilled in the art would understand that the cDNAsequence of NANOG as described above would be equivalent to NANOG mRNA,and can be used for the generation of RNA capable of expressing NANOG.

LIN28 is a conserved cytoplasmic protein with an unusual pairing ofRNA-binding motifs: a cold shock domain and a pair of retroviral-typeCCHC zinc fingers. In mammals, it is abundant in diverse types ofundifferentiated cells. In pluripotent mammalian cells, LIN28 isobserved in RNase-sensitive complexes with Poly(A)-Binding Protein, andin polysomal fractions of sucrose gradients, suggesting it is associatedwith translating mRNAs.

Preferably, “LIN28 protein” or simply “LIN28” relates to human LIN28 andpreferably comprises an amino acid sequence encoded by the nucleic acidaccording to SEQ ID NO: 7, preferably the amino acid sequence accordingto SEQ ID NO: 8. One skilled in the art would understand that the cDNAsequence of LIN28 as described above would be equivalent to LIN28 mRNA,and can be used for the generation of RNA capable of expressing LIN28.

Krueppel-like factor (KLF4) is a zinc-finger transcription factor, whichis strongly expressed in postmitotic epithelial cells of differenttissues, e.g. the colon, the stomach and the skin. KLF4 is essential forthe terminal differentiation of these cells and involved in the cellcycle regulation.

Preferably, “KLF4 protein” or simply “KLF4” relates to human KLF4 andpreferably comprises an amino acid sequence encoded by the nucleic acidaccording to SEQ ID NO: 9, preferably the amino acid sequence accordingto SEQ ID NO: 10. One skilled in the art would understand that the cDNAsequence of KLF4 as described above would be equivalent to KLF4 mRNA,and can be used for the generation of RNA capable of expressing KLF4.

MYC (cMYC) is a protooncogene, which is overexpressed in a wide range ofhuman cancers. When it is specifically-mutated, or overexpressed, itincreases cell proliferation and functions as an oncogene. MYC geneencodes for a transcription factor that regulates expression of 15% ofall genes through binding on Enhancer Box sequences (E-boxes) andrecruiting histone acetyltransferases (HATs). MYC belongs to MYC familyof transcription factors, which also includes N-MYC and L-MYC genes.MYC-family transcription factors contain the bHLH/LZ (basicHelix-Loop-Helix Leucine Zipper) domain

Preferably, “cMYC protein” or simply “cMYC” relates to human cMYC andpreferably comprises an amino acid sequence encoded by the nucleic acidaccording to SEQ ID NO: 11, preferably the amino acid sequence accordingto SEQ ID NO: 12. One skilled in the art would understand that the cDNAsequence of cMYC as described above would be equivalent to cMYC mRNA,and can be used for the generation of RNA capable of expressing cMYC.

A reference herein to specific factors such as OCT4, SOX2, NANOG, LIN28,KLF4 or c-MYC or to specific sequences thereof is to be understood so asto also include all variants of these specific factors or the specificsequences thereof as described herein. In particular, it is to beunderstood so as to also include all splice variants,posttranslationally modified variants, conformations, isoforms andspecies homologs of these specific factors/sequences which are naturallyexpressed by cells.

According to the present invention, the term “peptide” comprises oligo-and polypeptides and refers to substances comprising two or more,preferably 3 or more, preferably 4 or more, preferably 6 or more,preferably 8 or more, preferably 10 or more, preferably 13 or more,preferably 16 more, preferably 21 or more and up to preferably 8, 10,20, 30, 40 or 50, in particular 100 amino acids joined covalently bypeptide bonds. The term “protein” refers to large peptides, preferablyto peptides with more than 100 amino acid residues, but in general theterms “peptides” and “proteins” are synonyms and are usedinterchangeably herein.

Proteins and peptides described according to the invention may beisolated from biological samples such as tissue or cell homogenates andmay also be expressed recombinantly in a multiplicity of pro- oreukaryotic expression systems.

For the purposes of the present invention, “variants” of a protein orpeptide or of an amino acid sequence comprise amino acid insertionvariants, amino acid deletion variants and/or amino acid substitutionvariants.

Amino acid insertion variants comprise amino- and/or carboxy-terminalfusions and also insertions of single or two or more amino acids in aparticular amino acid sequence. In the case of amino acid sequencevariants having an insertion, one or more amino acid residues areinserted into a particular site in an amino acid sequence, althoughrandom insertion with appropriate screening of the resulting product isalso possible.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example: (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid.

Substitutions typically may be made within groups (a)-(d). In addition,glycine and proline may be substituted for one another based on theirability to disrupt α-helices. Some preferred substitutions may be madeamong the following groups: (i) S and T; (ii) P and G; and (iii) A, V, Land I. Given the known genetic code, and recombinant and synthetic DNAtechniques, the skilled scientist readily can construct DNAs encodingthe conservative amino acid variants.

Preferably the degree of similarity, preferably identity between aspecific amino acid sequence described herein and an amino acid sequencewhich is a variant of said specific amino acid sequence will be at least70%, preferably at least 80%, preferably at least 85%, even morepreferably at least 90% or most preferably at least 95%, 96%, 97%, 98%or 99%. The degree of similarity or identity is given preferably for aregion of at least about 20, at least about 40, at least about 60, atleast about 80, at least about 100, at least about 120, at least about140, at least about 160, at least about 200 or 250 amino acids. Inpreferred embodiments, the degree of similarity or identity is given forthe entire length of the reference amino acid sequence.

The amino acid variants described above may be readily prepared with theaid of known peptide synthesis techniques such as, for example, by solidphase synthesis (Merrifield, 1964) and similar methods or by recombinantDNA manipulation. The manipulation of DNA sequences for preparingproteins and peptides having substitutions, insertions or deletions, isdescribed in detail in Sambrook et al. (1989), for example.

According to the invention, “variants” of proteins and peptides alsocomprise single or multiple substitutions, deletions and/or additions ofany molecules associated with the protein or peptide, such ascarbohydrates, lipids and/or proteins or peptides. The term “variants”also extends to all functional chemical equivalents of said proteins andpeptides.

According to the invention, a variant of a protein or peptide preferablyhas a functional property of the protein or peptide from which it hasbeen derived. Such functional properties are described above for OCT4,SOX2, NANOG, LIN28, KLF4 and c-MYC, respectively. Preferably, a variantof a protein or peptide has the same property in reprogramming an animaldifferentiated cell as the protein or peptide from which it has beenderived. Preferably, the variant induces or enhances reprogramming of ananimal differentiated cell.

The methods of the present invention can be used to effectde-differentiation of any type of somatic cell. Cells that may be usedinclude cells that can be de-differentiated or reprogrammed by themethods of the present invention, in particular cells that are fully orpartially differentiated, more preferably terminally differentiated.Preferably, the somatic cell is a diploid cell derived frompre-embryonic, embryonic, fetal, and post-natal multi-cellularorganisms. Examples of cells that may be used include but are notlimited to fibroblasts, such as fetal and neonatal fibroblasts or adultfibroblasts, keratinocytes, in particular primary keratinocytes, morepreferably keratinocytes derived from hair, B cells, T cells, dendriticcells, adipose cells, epithelial cells, epidermal cells, chondrocytes,cumulus cells, neural cells, glial cells, astrocytes, cardiac cells,esophageal cells, muscle cells, melanocytes, hematopoietic cells,osteocytes, macrophages, monocytes, and mononuclear cells.

The cells with which the methods of the invention can be used can be ofany animal species; e.g., mammals and rodents. Examples of mammaliancells that can be de-differentiated and re-differentiated by the presentinvention include but are not limited to human and non-human primatecells. Primate cells with which the invention may be performed includebut are not limited to cells of humans, chimpanzees, baboons, cynomolgusmonkeys, and any other New or Old World monkeys. Rodent cells with whichthe invention may be performed include but are not limited to mouse,rat, guinea pig, hamster and gerbil cells.

The term “organism” according to the invention relates to any biologicalunit that is capable of multiplying or transmitting genetic material andcomprises plants and animals, and microorganisms such as bacteria,yeasts, fungi and viruses. The term “organism” includes but is notlimited to a human being, a nonhuman primate or another animal, inparticular a mammal such as a cow, horse, pig, sheep, goat, dog, cat ora rodent such as a mouse and rat. In a particularly preferredembodiment, the organism is a human being.

De-differentiated cells prepared according to the present invention areexpected to display many of the same requirements as pluripotent stemcells and can be expanded and maintained under conditions used forembryonic stem cells, e.g. ES cell medium or any medium that supportsgrowth of the embryonic cells. Embryonic stem cells retain theirpluripotency in vitro when maintained on inactivated fetal fibroblastssuch as irradiated mouse embryonic fibroblasts or human fibroblasts(e.g., human foreskin fibroblasts, human skin fibroblasts, humanendometrial fibroblasts, human oviductal fibroblasts) in culture. In oneembodiment, the human feeder cells may be autologous feeder cellsderived from the same culture of reprogrammed cells by directdifferentiation.

Furthermore, human embryonic stem cells can successfully be propagatedon Matrigel in a medium conditioned by mouse fetal fibroblasts. Humanstem cells can be grown in culture for extended period of time andremain undifferentiated under specific culture conditions.

In certain embodiments, the cell culture conditions may includecontacting the cells with factors that can inhibit differentiation orotherwise potentiate de-differentiation of cells, e.g., prevent thedifferentiation of cells into non-ES cells, trophectoderm or other celltypes.

De-differentiated cells prepared according to the present invention canbe evaluated by methods including monitoring changes in the cells'phenotype and characterizing their gene and protein expression. Geneexpression can be determined by RT-PCR, and translation products can bedetermined by immunocytochemistry and Western blotting. In particular,de-differentiated cells can be characterized to determine the pattern ofgene expression and whether the reprogrammed cells display a pattern ofgene expression similar to the expression pattern expected ofundifferentiated, pluripotent control cells such as embryonic stem cellsusing techniques well known in the art including transcriptomics.

The expression of the following genes of de-differentiated cells can beassessed in this respect: OCT4, NANOG, growth and differentiation factor3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4(FGF4), embryonic cell-specific gene 1 (ESG1), developmentalpluripotency-associated 2 (DPPA2), DPPA4, telomerase reversetranscriptase (TERT), embryonic antigen-3 (SSEA-3), SSEA-4,tumor-related antigen-1-60 (TRA-1-60), TRA-1-81, and TRA-2-49/6E

The undifferentiated or embryonic stem cells to which the reprogrammedcells may be compared may be from the same species as the differentiatedsomatic cells. Alternatively, the undifferentiated or embryonic stemcells to which the reprogrammed cells may be compared may be from adifferent species as the differentiated somatic cells.

In some embodiments, a similarity in gene expression pattern existsbetween a reprogrammed cell and an undifferentiated cell, e.g.,embryonic stem cell, if certain genes specifically expressed in anundifferentiated cell are also expressed in the reprogrammed cell. Forexample, certain genes, e.g., telomerase, that are typicallyundetectable in differentiated somatic cells may be used to monitor theextent of reprogramming. Likewise, for certain genes, the absence ofexpression may be used to assess the extent of reprogramming.

Self-renewing capacity, marked by induction of telomerase activity, isanother characteristic of stem cells that can be monitored inde-differentiated cells.

Karyotypic analysis may be performed by means of chromosome spreads frommitotic cells, spectral karyotyping, assays of telomere length, totalgenomic hybridization, or other techniques well known in the art.

Using the present invention, RNA encoding appropriate factors isincorporated into one or more somatic cells, e.g. by electroporation.After incorporation, cells are preferably cultured using conditions thatsupport maintenance of de-differentiated cells (i.e. stem cell cultureconditions). The de-differentiated cells can then be expanded andinduced to re-differentiate into different type of somatic cells thatare needed for cell therapy. De-differentiated cells obtained accordingto the present invention can be induced to differentiate into one ormore desired somatic cell types in vitro or in vivo.

Preferably, the de-differentiated cells obtained according to thepresent invention may give rise to cells from any of three embryonicgerm layers, i.e., endoderm, mesoderm, and ectoderm. For example, thede-differentiated cells may differentiate into skeletal muscle,skeleton, dermis of skin, connective tissue, urogenital system, heart,blood (lymph cells), and spleen (mesoderm); stomach, colon, liver,pancreas, urinary bladder; lining of urethra, epithelial parts oftrachea, lungs, pharynx, thyroid, parathyroid, intestine (endoderm); orcentral nervous system, retina and lens, cranial and sensory, gangliaand nerves, pigment cells, head connective tissue, epidermis, hair,mammary glands (ectoderm). The de-differentiated cells obtainedaccording to the present invention can be re-differentiated in vitro orin vivo using techniques known in the art.

In one embodiment of the present invention, the reprogrammed cellsresulting from the methods of this invention are used to producedifferentiated progeny. Thus, in one aspect, the present inventionprovides a method for producing differentiated cells, comprising: (i)obtaining reprogrammed cells using the methods of this invention; and(ii) inducing differentiation of the reprogrammed cells to producedifferentiated cells. Step (ii) can be performed in vivo or in vitro.Furthermore, differentiation can be induced through the presence ofappropriate differentiation factors which can either be added or arepresent in situ, e.g. in a body, organ or tissue into which thereprogrammed cells have been introduced. The differentiated cells can beused to derive cells, tissues and/or organs which are advantageouslyused in the area of cell, tissue, and/or organ transplantation. Ifdesired, genetic modifications can be introduced, for example, intosomatic cells prior to reprogramming. The differentiated cells of thepresent invention preferably do not possess the pluripotency of anembryonic stem cell, or an embryonic germ cell, and are, in essence,tissue-specific partially or fully differentiated cells.

One advantage of the methods of the present invention is that thereprogrammed cells obtained by the present invention can bedifferentiated without prior selection or purification or establishmentof a cell line. Accordingly in certain embodiments, a heterogeneouspopulation of cells comprising reprogrammed cells are differentiatedinto a desired cell type. In one embodiment, a mixture of cells obtainedfrom the methods of the present invention is exposed to one or moredifferentiation factors and cultured in vitro.

Methods of differentiating reprogrammed cells obtained by the methodsdisclosed herein may comprise a step of permeabilization of thereprogrammed cell. For example, cells generated by the reprogrammingtechniques described herein, or alternatively a heterogeneous mixture ofcells comprising reprogrammed cells, may be permeabilized beforeexposure to one or more differentiation factors or cell extract or otherpreparation comprising differentiation factors.

For example, differentiated cells may be obtained by culturingundifferentiated reprogrammed cells in the presence of at least onedifferentiation factor and selecting differentiated cells from theculture. Selection of differentiated cells may be based on phenotype,such as the expression of certain cell markers present on differentiatedcells, or by functional assays (e.g., the ability to perform one or morefunctions of a particular differentiated cell type).

In another embodiment, the cells reprogrammed according to the presentinvention are genetically modified through the addition, deletion, ormodification of their DNA sequence(s).

The reprogrammed or de-differentiated cells prepared according to thepresent invention or cells derived from the reprogrammed orde-differentiated cells are useful in research and in therapy.Reprogrammed pluripotent cells may be differentiated into any of thecells in the body including, without limitation, skin, cartilage, boneskeletal muscle, cardiac muscle, renal, hepatic, blood and bloodforming, vascular precursor and vascular endothelial, pancreatic beta,neurons, glia, retinal, neuronal, intestinal, lung, and liver cells.

The reprogrammed cells are useful for regenerative/reparative therapyand may be transplanted into a patient in need thereof. In oneembodiment, the cells are autologous with the patient.

The reprogrammed cells provided in accordance with the present inventionmay be used, for example, in therapeutic strategies in the treatment ofcardiac, neurological, endocrinological, vascular, retinal,dermatological, muscular-skeletal disorders, and other diseases.

For example, and not intended as a limitation, the reprogrammed cells ofthe present invention can be used to replenish cells in animals whosenatural cells have been depleted due to age or ablation therapy such ascancer radiotherapy and chemotherapy. In another non-limiting example,the reprogrammed cells of the present invention are useful in organregeneration and tissue repair. In one embodiment of the presentinvention, reprogrammed cells can be used to reinvigorate damaged muscletissue including dystrophic muscles and muscles damaged by ischemicevents such as myocardial infarcts. In another embodiment of the presentinvention, the reprogrammed cells disclosed herein can be used toameliorate scarring in animals, including humans, following a traumaticinjury or surgery. In this embodiment, the reprogrammed cells of thepresent invention are administered systemically, such as intravenously,and migrate to the site of the freshly traumatized tissue recruited bycirculating cytokines secreted by the damaged cells. In anotherembodiment of the present invention, the reprogrammed cells can beadministered locally to a treatment site in need of repair orregeneration.

The term “patient” means according to the invention a human being, anonhuman primate or another animal, in particular a mammal such as acow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse andrat. In a particularly preferred embodiment, the patient is a humanbeing.

The terms “a” and “an” and “the” and similar referents used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein is merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illustrate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

EXAMPLES Example 1: Production of IVT RNA

The first step in the production of IVT RNA comprises linearization of aplasmid containing the coding sequence for a particular factor andhaving an SP6 promoter or T7 promoter before the start codon, startingfrom which an in vitro transcription is possible. To this end,restriction enzymes are used, for example. Following linearization, theenzyme is inactivated by phenol-chloroform precipitation and removed.For this, an isovolume of a mixture of phenol and chloroform is addedand mixed thoroughly.

Brief centrifugation at 10 000×g provides separation into a lowerorganic phase and an upper aqueous phase, which contains the DNA. Thelatter is transferred to a new reaction vessel. Then the aqueous phaseis mixed with an isovolume of pure chloroform, to remove any phenolresidues. After centrifugation, the aqueous phase is removed andprecipitated for 2 h by adding two isovolumes of ethanol and 10% v/v 3Msodium acetate pH 4.5 at −20° C. The DNA is sedimented by centrifugationfor 45 min at 10 000×g at 4° C., washed with 70% ethanol for removal ofsalts, and is taken up in a suitable volume of RNAse-free water. Gelelectrophoresis is used to verify that linearization was successful andcomplete. The concentration of the DNA is determined photometrically at260 nm. For determination of the purity of the DNA, in addition theoptical density is measured at 280 nm to obtain the OD260/280 ratios.

10 μg of linearized DNA is used for the in vitro transcription. Forthis, 40 μl dNTPs, with 4/5 of the dGTP additionally provided with aCap-structure, 10 μl 10× buffer, 20 μl dTT and 10 μl of T7 or SP6polymerase are incubated for 2 h at 37° C. The polymerases bind to theirT7 or SP6 recognition sequences, which are located 5′ from the ORF thatis to be transcribed, and synthesize the complementary RNA strand.

The IVT RNA is purified with the MegaClear Kit. For this, it is taken upin a binding buffer concentrate, containing the necessary salts foroptimal binding of the RNA to the silica membrane. Addition of ethanolremoves water from the RNA hydration shell. The mixture is loaded in asilica column and centrifuged at 10 000×g for 2 min. The RNA binds tothe column, whereas impurities, e.g. enzyme residues, are washed away.After several washing steps, the purified RNA is eluted. The elutionbuffer is preheated to 95° C. to make elution more efficient.

Quality control and quantification are performed by gel electrophoresisand by photometry.

Example 2: Electroporation of Cells

The principle of electroporation is based on disturbing thetransmembrane potential of the cells by a brief current pulse. Thealteration of the transmembrane potential by an external stimulus isdescribed by the following equation:

ΔV _(m) =fE _(ext) r cos ϕ

V_(m) is the transmembrane potential and f is a form factor, whichdescribes the influence of the cell on the extracellular fielddistribution. fE_(ext) describes the applied electric field, r the cellradius and ϕ the angle to the externally applied electric field. Factorf is often given as 1.5, though it depends on many other factors. Theelectroporation of the cells is successful if the applied electric fieldexceeds the capacity of the cell membrane, i.e. ΔV_(m) is greater than athreshold value ΔV_(G), given as 1V (Kinosita, K., Jr. and Tsong, T. Y.(1977) Nature 268, 438-441). Since construction of the cell membrane asa bilayer is a feature that is common to eukaryotic cells, this valueshows little variation for different cell lines.

Through dielectric breakdown of the transmembrane potential, transientlyhydrophilic pores are formed, through which water penetrates into thecell, transporting molecules e.g. nucleic acids into the cells (Weaver,J. C. (1995) Methods Mol. Biol. 55, 3-28; Neumann, E. et al. (1999)Bioelectrochem. Bioenerg. 48, 3-16).

Prior to electroporation, the adherent cells used are cultivated up tosemi-confluence, washed with PBS and detached from the cell cultureflasks with trypsin. The cells are transferred to medium with 10% FCS(fetal calf serum) and centrifuged for 8 min at 500×g. The pellet isresuspended in the serum free medium X-Vivo and centrifuged again for 8min at 500×g. This washing operation is carried out two more times inorder to remove residues of FCS, which would interfere with subsequentelectroporation.

After washing, the cells are adjusted in 250 μl to the desired celldensity, transferred to the electroporation cuvettes and placed on ice.After adding the appropriate amount of in vitro transcribed RNA andstirring thoroughly, electroporation is carried out at 200V and 250 μF.Then the cells are transferred immediately to the adequate nutrientmedium for incubation.

According to the invention, it was possible to transfect cell lines withan efficiency of up to 90% or even more.

Moreover, an influence of cell count on transfection efficiency could beexcluded in a range between 2×10⁶ and 2×10⁷ cells. The electroporationconditions also do not have a decisive influence on transfectionefficiency in the range tested

Stability of the RNA was demonstrated over a period of 24 h, whereas theprotein can be detected in the cells over a period of 48-72 h, dependingon its half-life.

It could be shown that there is a direct dependency between the amountof transfected RNA and the amount of protein available.

Example 3: Stability of Proteins Expressed by Transfected RNA

We next examined for how long proteins with different halftimesfollowing transfer of IVT RNA can be stably detected in cells. 786-0cells were transfected with 20 μg eGFP IVT RNA and 2dGFP IVT RNA,respectively, and the fluorescence intensity was measured in the timecourse of 3 h to 120 h. The eGFP protein has a halftime of 16 h, whilethe halftime of the destabilized variant 2dGFP due to the integration ofa PEST amino acid sequence effecting protein degradation is reduced to 2h (Clontech, 1998). The experiment showed that already after 4 h asubstantial amount of translated protein was detectable which furtherincreased until 24 h after transfection. The amount of eGFP proteinremains relatively constant for more than 120 h. Even the destabilized2dGFP shows stable protein expression for 48 h (FIG. 1). In order toinduce protein expression of 2dGFP which is stable over a long period oftime RNA can be transfected every 48 h.

Example 4: Examination of Effects Due to the Methodology Following RNAElectroporation

We next examined which unspecific effects are induced by electroporationand introduction of single stranded RNA into cells. These effects couldsuperimpose the cell differentiation induced by a specific gene producttransferred as IVT RNA. 2×10⁷ 786-0 cells were transfected with 20 μgeGFP IVT RNA and were cultivated for further 8 h, 24 h and 72 h. Acomparison with untransfected cells did not show a change in growthbehaviour and did not show an intensified apoptosis. The portion ofliving cells following transfection in each case was about 95%.

In a further step, RNA was extracted from the cells and used forpreparing probes. The probes were hybridized on a cDNA microarray chipwith several hundreds of genes. This was followed by a basic evaluationusing the program ImaGene software version 4.1 (BioDiscovery, LosAngeles, Calif.). Impurities on the array which were visible to thenaked eye were masked manually. Following normalization using thecontrol genes which were also spotted on the array, the relativeexpression levels compared to the reference were obtained. The number ofsignificantly regulated genes is shown in Table 1.

TABLE 1 Number of significantly regulated genes in 2 × 10⁷ 786-0 cellswhich were transfected with 20 μg eGFP IVT RNA compared tonon-transfected control cells. Only a regulation of > 2 and 0.5,respectively, was considered a significant change. Number of Number ofNumber of Factor of regulated genes regulated genes regulated genesregulation after 8 h after 24 h after 72 h >2 10 (0.87%) 0 0 <0.5 48(4.16%) 15 (1.3%) 0

The number of significantly regulates genes was moderate and decreasedover time. 24 h after electroporation and introduction of singlestranded RNA into the cells only 15 genes (1,3%) are stilldifferentially expressed. The eGFP-specific regulation of these geneswas excluded by means of an additional analysis of cells which weretransfected using irrelevant IVT RNA. Accordingly, the dysfunction ofthe transcriptome of the cells in terms of an unspecific regulation ofgenes which is inherently caused by the methodology is low.Reprogramming of cells by transfection with IVT RNA thus is expected tobe not affected by difficulties which are inherent to the methodology oronly to a very small extent.

Example 5: Reprogramming of Cells by Means of Introduction of RNA Codingfor Transcription Factors

To examine whether the introduction of genes can be used for changingthe cellular program, we have analyzed the effect of the transfection ofthe oncogenic transcription factors SYT-SSX1 and SYT-SSX2 which resultfrom the translocation t(X;18)(p11.2;q11.2) (Clark et al., 1994; Crew etal., 1995) and are detectable in more than 90% of the synovial sarcomas(Sreekantaiah et al., 1994). The molecular changes caused by thetransfection of SYT-SSX1 and SYT-SSX2 IVT RNA were analyzed usingAffymetrix oligonucleotide microarrays. The different constructs eachwere transfected in triplicate. Transfection with eGFP was performed inorder to be able to examine the transfection efficiency in thefluorescence microscope. After 8 h a transfection efficiency of morethan 95% was determined (data not shown). The cells were harvested after8 h, 24 h and 72 h, respectively, and the RNA was extracted. Foranalysing the molecular changes in the cells following RNA transfer ofthe listed genes we used Affymetrix oligonucleotide microarrays whichenabled the simultaneous examination of changes in expression of 22,000genes.

For analysing cells transfected with SYT-SSX2 and eGFP two human genomeU133A arrays were hybridized at 8 h and 24 h in each case. SSX2 andSYT-SSX1 were examined in single determinations at 8 h and 24 h.Evaluation of the data was done using the Software Microarray Suite 5.0as well as ArrayAssist (FIG. 2). The number of the significantlyregulated genes in the comparison of the duplicates among each other waszero. This resulted in a sufficient confidence with respect to thereproducibility of the results such that also the microarray results ofthe transfectants which were only represented by single determinations(SYT-SSX1 and SSX2) could be included in the analysis.

For determining the genes which are significantly regulated by SYT-SSX2,the expression values of the eGFP transfected cells were taken as basisand the expression pattern of the SSX2, SYT-SSX1 and SYT-SSX2 IVT RNAtransfected cells were compared thereto (FIG. 3). Evaluation of the datawas performed using the programs Microarray Suite 5.0 and ArrayAssist.FIG. 3a exemplarily demonstrates the differential expression of genes bySYT-SSX2.

For evaluation, a regulation by at least a factor of two was taken as abasis which corresponds to the range of sensitivity of the system. Avalue of p=5% was considered as a criterion of significance. Under theseconditions, 185 genes could be detected in the SYT-SSX2 transfectedcells after 8 h and 218 genes could be detected after 24 h. The portionof genes which are regulated after 8 h as well as after 24 h is 41.3%(FIG. 3b ).

The regulated genes can be assigned to different groups on the basis oftheir function. These are, for example, growth factors, neuronal genes,tumor associated genes, collagens, as well as those which are involvedin the processes of signal transduction, cell adhesion, cell developmentand cell differentiation as well as in the regulation of the cell cycle.

In this respect it is noticeable that the portion of overexpressed genesis significantly higher than the portion of suppressed genes. The geneswhich are regulated by SYT-SSX1 are >95% identical to those which arealso regulated by SYT-SSX2.

The differential expression in vivo of the analyzed genes could beclearly confirmed in synovial sarcomas. The increased expression of BMP7and EPHA4 was already described in other studies (Nagayama et al.,2002). Overexpression in synovial sarcomas was also detectable forFGFR4, p5′7, BMP5 and PGF.

These studies show that the transfer of RNA of transcription factors canbe successfully used to change differentiation of cells.

Example 6: Reprogramming of Cells by Means of Introduction of RNA Codingfor Transcription Factor Cocktails

In-vitro translated mRNA (IVT-RNA) encoding the TFs cocktail OCT4, SOX2,KLF4 and c-MYC (OSKM) or OCT4, SOX2, LIN28 and NANOG (OSLN) waselectro-transferred into the cytoplasm of human or murine fibroblasts.

In a first set of experiments, we optimized the electroporationparameters using IVT-RNA encoding eGFP for human newborn foreskinfibroblasts (CCD1079Sk) and mouse embryonic fibroblasts (MEF).

As a general measure to increase the stability of the IVT-RNA constructsand the protein translation, the nucleotide sequence of the TFs has beencodon optimized to increase the GC-content and to enhance translation inhuman or mouse cells. Since the expression efficiency and stability ofIVT-RNA is mainly dependent on the 5′-CAP structure, we evaluated theeffect of three different 5′-Cap-Structures (FIG. 4) that are wellestablished in our lab on the expression of luciferase inCCD1079Sk-cells.

We found that the efficiency of electroporation is consistently higherthan 90%, which is higher than the retroviral transduction efficienciespublished by others (Takahasi et al., 2006; Takahasi et al., 2007).Especially, one has to consider that an infection efficiency of 80% forone retroviral vector means that cotranduction efficiencies for all 4required vectors will be lower (0.8⁴=0.4). Our approach, theco-electroporation of the 4 TFs will ensure the transfer of all 4factors to more than 90% of the cells.

We observed that the highest efficiency is achieved when CCD1079SK cellsor MEFs are electroporated with 300 μF/250V or 500 μF/250V respectivelyregarding the mean fluorescence of eGFP (which corresponds to thehighest expression level)(FIG. 5).

We furthermore found that the IVT-mRNA with D1 cap structure displayedthe highest and most stable expression of luciferase in CCD1079Sk cells(FIG. 6) and was therefore chosen for the subsequent experiments. TheIVT-mRNA with D2 cap structure displayed higher and more stableexpression of luciferase than IVT-mRNA with ARCA cap structure.

Next we examined the intracellular amount of the electroporated RNA andexpression levels of these exogenous mRNA constructs encoding the sixTFs SOX2, OCT4, KLF4, cMYC, NANOG, and LIN28. Oligonucleotides specificfor the codon-optimized constructs were used in qRT-PCR studies. In thesame set of experiments we determined the half-life of the IVT-RNA andthe encoded proteins in time course experiments.

We found that:

(i) high levels of IVT-RNA can be detected by qRT-PCR after 24 h inelectroporated CCD1079Sk cells (FIG. 7),

(ii) the IVT-RNA of all six transcription factors is well detectableeven 168 h post electroporation (FIG. 7),

(iii) the IVT-RNA encoding the four TFs SOX2, OCT4, KLF4, and cMYC(OSKM) are translated to high protein levels 24 h post electroporationas monitored by western blot analysis (FIG. 8),

(iv) OCT4—together with SOX2 the most important TF for reprogramming(Huangfu et al., 2008)—was expressed for 72 h (in CCD1079Sk cells) to 96h (in MEFs) at levels similar to or higher levels as in NTERA cells, anembryonic carcinoma cell line—SOX2 expression was detectable for 48 h(in CCD1079Sk cells) to 72 h (in MEFs) (FIG. 8) and

(v) MYC and KLF4 were expressed at least 24 h (in CCD1079Sk cells) and48 h (in MEFs). For both TF short half-lives are published (Chen et al.,2005; Sears et al., 2000) (FIG. 8).

Since it is well established that all mammalian pluripotent stem cellsexpress alkaline phosphatase (AP) activity and that AP is an earlymarker of the reprogramming process (Pera et al., 2000; Brambrink etal., 2008, Cell Stem Cell. 7, 151-159; O'Connor et al., 2008), weinvestigated the induction of AP expression upon a singleelectroporation of IVT-RNA encoding the four TFs OSKM (15 μg each TF).

We found that 10 days post electroporation about 6% of the cells werestained positive for AP as revealed by FACS analysis (FIG. 9). Thesedata match to data published recently showing that 3 days expression ofthe four TFs OSKM in a doxocyclin-inducible system leads to about 5%AP-positive cells (Brambrink et al., 2008). As mentioned above, a singleelectroporation leads also to an expression of 3-4 days.

Nevertheless a single electroporation was not sufficient to induce thegrowth of iPS colonies. This is in accordance to recently published datashowing that the induction of AP is reversible and TFs need to beexpressed at least 12 days to complete the reprogramming process(Brambrick et al, 2008). On the basis of the time course experiments(FIG. 8) CCD1079Sk cells and MEFs were therefore repeatedlyelectroporated every 48 h, which means that 6 electroporations arerequired to ensure at least 12 days of TF expression.

After different time points we stained the cells for the earlyreprogramming marker AP and analyzed by fluorescence microscopy.Additionally we isolated total RNA from cells just prior eachelectroporation and evaluated mRNA-expression of endogenous human andmurine ES cell markers by quantitative real-time PCR. We included theHDAC-inhibitor VPA in our study because it has been demonstrated thatVPA enhances the reprogramming efficiency (Huangfu et al., 2008).

We found that:

(i) cells were reproducible stained positive for AP. AP was induced evenat earlier time points as analyzed in the first experiment (4 to 7days). The amount of IVT-RNA resulting in AP positivity reached from asless as 1.25 μg per TF to 5 μg per TF (FIGS. 10 and 11),

(ii) the addition of VPA greatly increases the percentage of AP-positiveMEFs electroporated with IVT-RNA encoding the four TFs OSKM (FIG. 11),

(iii) human and murine ES cell markers that further underline thereprogramming process have been induced: endogenous human OCT4 (FIG.12), human and murine TERT (telomerase reverse transcriptase) (FIG.12-14), human GDF3 (growth differentiation factor 3) (FIGS. 12 and 13)and human DPPA4 (developmental pluripotency associated 4) (FIG. 13). Asfor AP addition of VPA increased the induction of human and murine EScell marker (FIG. 12-14) and

(iv) repetitive electroporation is associated with a loss of cellviability which became apparent only after the second electroporation.The viability further decreased with every following electroporation.

The addition of previously not electroporated cells (serving as“carrier” cells) during the 4th and 6th electroporation turned out torescue the electroporated cells (FIG. 14A).

We found that a large number of cells remained viable after the lastelectroporation. This enabled us to plate these cells onto irradiatedMEF feeder cells. The outgrowth of pluripotent colonies from these cellsis still under investigation.

Taken together our data show that the electro-transfer of IVT-RNAencoding TFs regulating stem cell pluripotency into somatic cellssuccessfully initiates the reprogramming process. It is expected thatthe problem of limited viability of the cells will be improved byreducing the time of TF expression necessary to reprogram the cells orincreasing the survival of the cells during electroporation.

The following measures are expected to reduce the duration ofreprogramming:

(i) It has been recently published that keratinocytes are more rapidlyand more efficiently reprogrammed to pluripotent cells than fibroblasts(Aasen et al., 2008, Nat. Biotechnol. 26, 1276-84). Therefore, it isexpected that by using keratinocytes such as primary keratinocytes(“normal human epidermal keratinocytes; Promocell, Heidelberg, Germany)a reduced number of electroporations will be sufficient to cover therequired expression period.

(ii) It has recently been published that the expression of proteins thatare known to immortalize cells, hTERT and SV40 large-T antigen, enhancethe efficiency and pace of reprogramming (Park et al., 2008, NatureProtocols 3, 1180-1186; Mali et al., 2008). Therefore, addition ofIVT-RNA encoding such proteins such as IVT-RNA encoding codon optimizedlarge-T antigen to the TF-cocktail is expect to provide a beneficialeffect.

The following measures are expected to increase the survival of thecells:

(i) Voltage and capacity settings of electroporations used herein havebeen optimized for maximal expression levels after one electroporation.However, even milder conditions resulted in similar percentages oftransfected cells with slightly reduced expression levels as monitoredby the mean fluorescence of GFP (FIG. 4). Therefore, the settings shouldbe further optimized regarding survival after repeated electroporationswithout accepting transfection efficiency lower than 75%.

(ii) Our results have shown that human fibroblasts become and remain APpositive for at least 10 days after a single electroporation which istwice or three times longer than the expression of the exogenous TFs. Wenoted in our time course experiments that human fibroblast start torecover from electroporation induced damage after about 96 hours.Therefore we reason that the frequency of electroporations might beraised to 96 or 120 hours to allow a better recovery of the cellswithout impairing the reprogramming process.

(iii) Besides the modifications in electroporation conditions andfrequencies, an increased survival can also be obtained by inhibitingapoptosis by preventing upregulation of the pro-apoptotic protein p53.To this aim we will add the human papilloma virus 16 transformingprotein E6 (HPV-16 E6) to our TF-cocktail. E6 inhibits apoptosis byinducing the poly-ubiquitinylatuion and proteasomal degradation of p53and by interfering with other pro-apoptotic proteins (Bak, FADD,procaspase-8). Furthermore it induces the expression of hTERT andcooperates positively with c-MYC (Ristriani et al., 2008; Narisawa-Saito& Kiyono 2007).

(iv) In addition we will increase the half-life of the MYC protein byintroducing a stabilizing point mutation (Thr-58 to Ala-58) that isfound in Burkitt's lymphoma and has been described previously (Sears etal., 2000; Gregory & Hann 2000).

(v) We will functionally delete a destabilizing PEST domain in c-MYC inorder to further increase the half-life. PEST-domain deletions have beenshown to increase the stability of c-MYC (Gregory & Hann 2000).

1-36. (canceled)
 37. A RNA-transfected somatic cell population whichcomprises somatic cells subjected to electroporation, containing invitro transcribed mRNA encoding OCT4, SOX2, KLF4 and c-MYC, wherein themRNA comprises a 5′-Cap structure and wherein the transfected cellpopulation expresses alkaline phosphatase and expresses endogenous OCT4.38. The RNA-transfected somatic cell population in accordance with claim37, wherein the somatic cells are human cells.
 39. The RNA-transfectedsomatic cell population in accordance with claim 38, wherein the humancells are adult human dermal fibroblasts.
 40. The RNA transfectedsomatic cell population in accordance with claim 37, wherein the somaticcells are fibroblasts.
 41. The RNA transfected somatic cell populationin accordance with claim 37, which expresses endogenous OCT4 for atleast 7 days.
 42. The RNA-transfected somatic cell population inaccordance with claim 37, wherein the somatic cells are fibroblastsdeposited at American Type Culture Collection under Catalog No. CCL-186.43. The RNA-transfected somatic cell population in accordance with claim37, wherein the somatic cells are fibroblasts deposited at American TypeCulture Collection under Catalog No. CRL-2097.
 44. A method forproducing a RNA-transfected somatic cell population, which comprisesintroducing in vivo translated mRNA into somatic cells by repeatedelectroporation; the introduced mRNA encoding at least one transcriptionfactor which is a member of the group consisting of OCT4, SOX2, KLF4 andc-MYC.
 45. The method in accordance with claim 44, wherein theelectroporation is effected at 300 μF/250 V.
 46. The method inaccordance with claim 44, wherein the electroporation is effected at 500μF/500 V.